| Project/Area Number |
12558068
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 展開研究 |
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| Research Field |
環境保全
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| Research Institution | Tokyo University of Agriculture and Technology |
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Principal Investigator |
SATO Ryoichi Tokyo University of Agriculture and Technology, Graduate School of Bio-Applications & Systems-Engineering, Associate Professor, 大学院・生物システム応用科学研究科, 助教授 (30235428)
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| Co-Investigator(Kenkyū-buntansha) |
OGIWARA Katsutoshi Kubota Corporation, Technology Development Headquarters, Deputy Section Manager, 事業開発部BB?PT, 課長補佐(研究職)
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| Project Period (FY) |
2000 – 2002
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| Project Status |
Completed (Fiscal Year 2002)
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| Budget Amount *help |
¥13,200,000 (Direct Cost: ¥13,200,000)
Fiscal Year 2002: ¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 2001: ¥2,600,000 (Direct Cost: ¥2,600,000)
Fiscal Year 2000: ¥8,300,000 (Direct Cost: ¥8,300,000)
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| Keywords | Bacillus thuringiensis / Phage display / Directed evolution / Delta -endotoxin / 進化光学 |
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| Research Abstract |
We analyzed APN-binding sites using two methods that introduced blocking molecules on the surface of CrylAa toxin. Of seven monoclonal antibodies constructed against Cry1Aa toxin, 1B10 and 2C2 inhibited the binding of Cry1Aa toxin to BmAPN1, suggesting that their binding sites (epitopes) are located close to the BmAPNl binding site of the toxin. To determine the true epitopes of the antibodies, cysteine substitutions were introduced at 521Arg or 582Val on CrylAa and then a smaller blocking molecule, N-(9-acridinyl)maleimide (NAM), was covalently bound to the -SH of Arg521Cys and Val582Cys. The binding assay showed that both blocking antibodies bound the Val582Cys toxin, but not the Val582Cys-NAM toxin, suggesting that the epitopes of the two antibodies were located adjacent to the Val582Cys of Domain III. In addition, NAM covalently 〓ound to Val582Cys affected BmAPN1 binding to the Val582Cys toxin, but not BtR175 binding to it, and reduced the toxicity of Val582Cys toxin in Bombyx mori larvae. These results suggest that the BmAPN1 binding site on Cry1Aa is located near 582Val and that BmAPN1 functions as a receptor for Cry1Aa toxin in Bombyx mori larvae.
Next, we cloned 4 cDNAs of aminopeptidase N isoforms, showed primary structures from their deduced amino acid sequences, constructed antibodies against four each recombinant isoforms, and then clearly showed that Cry1Aa and Cry1Ab mainly bind to isoform 1 (BmAPN 1). Finally, we prepared 10^9 phages which express Cry1Aa toxin randomly mutated in domain 2 and 3. In addition, we succeeded to make a fundamental method to select high affinity toxin- expressing phages to cadherin-like receptor (BtR175) from the mixture of the phages using Cry1Aa and Cry1Ab as model materials to be expressed on the phages.
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